pharmacol rev 53:527–552, 2001 printed in u.s.a international … · of the receptors (jin et...

International Union of Pharmacology. XXV.Nomenclature and Classification of Adenosine Receptors

BERTIL B. FREDHOLM,1 ADRIAAN P. IJZERMAN, KENNETH A. JACOBSON, KARL-NORBERT KLOTZ, AND JOEL LINDEN

Department of Physiology and Pharmacology, Section of Molecular Neuropharmacology, Karolinska Institutet, Stockholm, Sweden (B.B.F.);Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden, The Netherlands (A.P.I.); Molecular Recognition Section, National

Institutes of Health, Bethesda, Maryland (K.A.J.); Institut fur Pharmakologie und Toxikologie, Universitat Wurzburg, Wurzburg, Germany(K.-N.K.); and Department of Physiology, Health Sciences Center, University of Virginia, Charlottesville, Virginia (J.L.)

This paper is available online at http://pharmrev.aspetjournals.org

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527

II. Molecular basis for receptor nomenclature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528III. Formation and levels of the endogenous agonist adenosine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529IV. Structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 530V. Gene structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531

VI. Binding sites as revealed by site-directed mutagenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532VII. Distributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535

VIII. Classification of adenosine receptors using pharmacological tools . . . . . . . . . . . . . . . . . . . . . . . . . . . 536IX. Signaling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541

A. G protein coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541B. Second messengers and signals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541C. Adenosine receptor-mediated changes in cell proliferation and in mitogen-activated protein

kinase activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 542D. Interactions with other receptor systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543

X. Receptor regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 544XI. Assay systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545

XII. Physiological roles—therapeutic potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547

Abstract——Four adenosine receptors have beencloned and characterized from several mammalianspecies. The receptors are named adenosine A1, A2A,A2B, and A3. The A2A and A2B receptors preferablyinteract with members of the Gs family of G proteinsand the A1 and A3 receptors with Gi/o proteins. How-ever, other G protein interactions have also been de-scribed. Adenosine is the preferred endogenous ago-nist at all these receptors, but inosine can also activatethe A3 receptor. The levels of adenosine seen underbasal conditions are sufficient to cause some activa-tion of all the receptors, at least where they are abun-dantly expressed. Adenosine levels during, e.g., isch-

emia can activate all receptors even when expressedin low abundance. Accordingly, experiments with re-ceptor antagonists and mice with targeted disruptionof adenosine A1, A2A, and A3 expression reveal roles forthese receptors under physiological and particularlypathophysiological conditions. There are pharmaco-logical tools that can be used to classify A1, A2A, and A3receptors but few drugs that interact selectively withA2B receptors. Testable models of the interaction ofthese drugs with their receptors have been generatedby site-directed mutagenesis and homology-basedmodelling. Both agonists and antagonists are beingdeveloped as potential drugs.

I. Introduction

The nomenclature and classification of adenosine re-ceptors has been covered in two publications by mem-

bers of a previous NC-IUPHAR subcommittee, whichwas devoted to “purinoceptors” (Fredholm et al., 1994a,1997). However, these two publications were progressreports and were not official documents of the NC-IUPHAR. They dealt with both adenosine receptors andP2 receptors. As a result of this work, separate subcom-mittees were set up for adenosine receptors, P2X recep-

1 Address for correspondence: Bertil B. Fredholm, Department ofPhysiology and Pharmacology, Section of Molecular Neuropharma-cology, Karolinska Institutet, 171 77 Stockholm, Sweden. E-mail:[email protected]

0031-6997/01/5304-527–552$3.00PHARMACOLOGICAL REVIEWS Vol. 53, No. 4Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 10401/947126Pharmacol Rev 53:527–552, 2001 Printed in U.S.A

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tors, and P2Y receptors. The present review will there-fore cover adenosine receptors only. The previouspublications contain the historical background and thiswill not be recapitulated.

The term adenosine receptor is used to denote thisgroup of receptors. First, use of the name adenosinefollows the recommendation of NC-IUPHAR that recep-tors be named after the preferred endogenous agonist.Second, and as discussed in the previous publications,the concept of adenosine receptors precedes the laterconcept of purinoceptors (P1 and P2) (Burnstock, 1978)by several years. The discovery by Drury and Szent-Gyorgyi (1929) that adenosine can influence severalbodily functions inspired much research interest; thepronounced cardiovascular effects of adenosine wereparticularly well investigated. Several adenosine ana-logs were synthesized, and examination of the dose-response relationships suggested the presence of specificadenosine receptors (Cobbin et al., 1974). The essen-tially competitive nature of the antagonism by methyl-xanthines, including caffeine and theophylline, of aden-osine effects in the heart (De Gubareff and Sleator,1965) and in the brain (Sattin and Rall, 1970) also sup-

ported the idea of adenosine receptors. Finally, as theterm P2 receptor has been superseded by the names P2Xand P2Y there is little room for the term P1 except whenreferring specifically to older publications.

There are four different adenosine receptors, denotedA1, A2A, A2B, and A3 (Table 1). This terminology is wellestablished and is coherent with the principles of recep-tor nomenclature adopted by NC-IUPHAR. Althoughthe primary basis for adenosine receptor nomenclatureis structural, historical reasons also played a role. Asdiscussed previously (Fredholm et al., 1994a), carefulpharmacological analysis first identified two sub-forms—A1 and A2 (van Calker et al., 1979; Londos et al.,1980a). Later pharmacological studies revealed that theA2 receptors, coupled to adenylyl cyclase, were heterog-enous, necessitating subdivision into A2A and A2B.

II. Molecular Basis for Receptor Nomenclature

Once the adenosine A1 receptor was defined usingbinding assays, several attempts were made to purifythe receptor. Despite considerable progress by severalgroups the receptor was never sufficiently pure to allow

TABLE 1Current adenosine receptor nomenclature

Receptor A1 A2A A2B A3

Receptor Code 2.1:ADO: 2.1:ADO: 2.1:ADO: 2.1:ADO:Previous names Ri A2a, Rs A2b, RsStructural

information7TM; human 326 aa,

P30542, chr 1q32.1, rat326 aa, P25099, mouse326 aa

7TM; human 410 aa, P29274,chr 22g11.2, rat 409 aa,P30543, mouse 409 aa,UO5672

7TM; human 328 aa,P29275, chr 17p11.2–12;rat 332 aa, P29276mouse 332 aa, UO5673

7TM; human 318 aa, P33765,chr 1p21–13, rat 320 aaP28647 (see comments),mouse 320 aa, AF069778

Selectiveagonists

CPA, CCPA, CHA CGS 21680, HENECA, CV-1808,CV-1674, ATL146e

None Cl-IB-MECA

Selectiveantagonists

DPCPXa 8-cyclopentyl-theophylline, WRC0571

selective: SCH 58261b,c

moderately selective:ZM241385,a KF 17387, CSC

MRS1754,d enprofylline,alloxazine (historical)

MRS 1220,e MRE 3008-F20,e

MRS 1191; MRS 1523

Tissue functions Bradycardia; inhibition oflipolysis; reducedglomerular filtration;tubero-glomerularfeedback, antinociception;reduction of sympatheticand parasympatheticactivity; presynapticinhibition; neuronalhyperpolarization;ischemic preconditioning

Regulation of sensorimotorintegration in basal ganglia;inhibition of plateletaggregation andpolymorphonuclear leukocytes;vasodilatation, protectionagainst ischemic damage,stimulation of sensory nerveactivity

Relaxation of smoothmuscle in vasculatureand intestine; inhibitionof monocyte andmacrophage function,stimulation of mast cellmediator release (somespecies)

Enhancement of mediatorrelease from mast cells(some species).Preconditioning (somespecies)

Phenotypes Tissue functions aboveconfirmed in knockoutmouse

Tissue functions aboveconfirmed in knockout mouse

Tissue functions aboveconfirmed in knockoutmouse

Comments Also cloned from dog,f cow,g

rabbiti,jAlso cloned from e.g. dog,h

guinea pig.k Variations instructure among humans

Alternative splicing in rat canyield product with 337 aa.l

Also cloned from sheepm 317aa, rabbitj 320 aa

a DPCPX and ZM241385 also have nanomolar affinity for the adenosine A2B receptor.b Dionisotti et al., 1997.c Ongini et al., 1999.d Kim et al., 2000.e Li et al., 1999; Baraldi et al., 2000b.f Libert et al., 1989, 1991.g Tucker et al., 1992.h Maenhaut et al., 1990.i Bhattacharya et al., 1993.j Hill et al., 1997.k Meng et al., 1994.l Sajjadi et al., 1996.m Linden et al., 1993.

528 FREDHOLM ET AL.

sequencing. Instead, the cloning of the first adenosinereceptors was serendipitous. Four novel members of theG protein-coupled receptor family were cloned from acanine thyroid library (Libert et al., 1989). Of these, oneturned out to be the adenosine A2A receptor (Maenhautet al., 1990), and another the adenosine A1 receptor(Libert et al., 1991). Once these first sequences wereobtained the same receptors were soon cloned from othermammals including human (Furlong et al., 1992; Libertet al., 1992; Townsend-Nicholson and Shine, 1992; Renand Stiles, 1995; Deckert et al., 1996; Peterfreund et al.,1996). In addition, the adenosine A2B receptor wascloned (Stehle et al., 1992; Jacobson et al., 1995). Moresurprisingly, a fourth adenosine receptor, denoted A3,was cloned, first as an orphan (Meyerhof et al., 1991),later as a bona fide methylxanthine-insensitive adeno-sine receptor in rat (Zhou et al., 1992), a xanthine-sensitive receptor in sheep (Linden et al., 1993), and apartially xanthine-sensitive receptor in humans (Sajjadiand Firestein, 1993; Salvatore et al., 1993; Linden,1994). Thus, a family of four adenosine receptors hasbeen cloned from several mammalian and nonmamma-lian species (see below). The current nomenclature issummarized in Table 1.

III. Formation and Levels of the EndogenousAgonist Adenosine

Adenosine is the main agonist at this receptor class,and this is the reason for the name. In addition, theadenosine metabolite inosine can activate at least someof the receptors (Jin et al., 1997; Fredholm et al., 2001),and there may be circumstances under which inosineprovides a larger activation than adenosine, but thisremains to be proven.

When given in very high amounts, adenosine can af-fect intracellular nucleotide pools and even provide asource of metabolizable energy. In addition, it was re-ported very recently that the human growth hormonesecretagogue receptor (GHS-R) also accepts adenosineas a highly potent endogenous agonist, in addition to theendogenous peptide GHS-R agonist, ghrelin (Smith etal., 2000; Tullin et al., 2000). However, most effects ofadenosine are due to activation of adenosine receptors.

Before we describe the activation of adenosine recep-tors under physiological conditions and hence the ac-tions of antagonists, the mechanisms regulating levels ofextracellular adenosine must be briefly presented. Un-der normal conditions, adenosine is continuously formedintracellularly as well as extracellularly. The intracellu-lar production is mediated either by an intracellular5�-nucleotidase, which dephosphorylates AMP (Schu-bert et al., 1979; Zimmermann et al., 1998), or by hydro-lysis of S-adenosyl-homocysteine (Broch and Ueland,1980). Adenosine generated intracellularly is trans-ported into the extracellular space mainly via specificbi-directional transporters through facilitated diffusion

that efficiently evens out the intra- and extracellularlevels of adenosine. In some tissues (e.g., kidney brush-border membranes) there is a concentrative nucleosidetransport protein capable of maintaining high adenosineconcentrations against a concentration gradient. Thesetransport proteins have been cloned and were termedENT1 and ENT2 (for the equilibrative transport pro-teins) and CNT1 and CNT2 (for the concentrative types)(e.g., Williams and Jarvis, 1991; Anderson et al., 1996;Baldwin et al., 1999). When the activity of transportersis decreased, e.g., by drugs or by reducing temperature,extracellular biologically active levels of adenosine in-crease (Dunwiddie and Diao, 2000). In view of the factthat several of the transporters are equilibrative, thismight seem to be a paradox. However, as discussedpreviously (e.g., Fredholm et al., 1994b), it must be re-membered that in tissue, some cells are net producers ofadenosine, and in these, intracellular levels risewhereas most cells are net eliminators of the nucleoside.

The dephosphorylation of extracellular AMP to aden-osine, mediated by ecto-5�-nucleotidase, is the last stepin the enzymatic chain that catalyzes the breakdown ofextracellular adenine nucleotides, such as ATP, to aden-osine. Ectonucleotidases include ectonucleoside triphos-phate diphosphohydrolases, including CD39, which canhydrolyze ATP or ADP, ectonucleotide pyrophosphatase/phosphodiesterases, alkaline phosphatases and 5�-nucleotidases such as CD73 (Zimmermann, 2000). Theseenzymes are essential for the nerve activity-dependentproduction of adenosine from released ATP under phys-iological conditions (Dunwiddie et al., 1997a; Zimmer-mann et al., 1998). The entire catalytic pathway is com-plete in a few hundred milliseconds, and the rate-limiting step seems to be the dephosphorylation of AMPto adenosine by ecto-5�-nucleotidase (Dunwiddie et al.,1997a). Recent data provide evidence for the presence ofsoluble 5�-nucleotidases of unknown structure that arereleased together with ATP from stimulated sympa-thetic nerve endings and participate in the extracellularhydrolysis of ATP to adenosine (Todorov et al., 1997). Instriatum, local application of a 5�-nucleotidase inhibitordose dependently decreases the normal levels of adeno-sine and thereby emphasizes the relevance of this en-zyme in vivo (Delaney and Geiger, 1998). Nonetheless,there is good evidence that intracellular formation ofadenosine is at least as important as adenosine forma-tion from breakdown of extracellular ATP (Lloyd et al.,1993; Lloyd and Fredholm, 1995). Intracellular forma-tion predominantly occurs as a consequence of activity ofintracellular 5�-nucleotidases, of which two forms, cN-Iand cN-II, have been cloned (Sala-Newby et al., 1999).These two enzymes may play different roles—cN-Ibreaking down AMP to adenosine and cN-II breakingdown IMP and GMP to inosine and guanosine, respec-tively (Sala-Newby et al., 2000).

When adenosine levels in the extracellular space arehigh, adenosine is transported into cells by means of

NOMENCLATURE AND CLASSIFICATION OF ADENOSINE RECEPTORS 529

transporters. It is then phosphorylated to AMP by aden-osine kinase (Km � approximately 100 nM; Spychala etal., 1996) or degraded to inosine by adenosine deami-nase, a process with a severalfold lower affinity (Km �20–100 �M; Arch and Newsholme, 1978; Lloyd andFredholm, 1995). In the heart, and probably also othertissues, hypoxia-induced inhibition of adenosine kinaseamplifies small changes in free myocardial AMP, result-ing in a major rise in adenosine (Decking et al., 1997).Adenosine deaminase, but not adenosine kinase, is alsopresent in the extracellular space (Lloyd and Fredholm,1995).

Adenosine can also be released into the extracellularspace after application of specific neurotransmitter li-gands. Glutamatergic agonists, such as NMDA or kai-nate, dose dependently increase adenosine levels (Car-swell et al., 1997; Delaney et al., 1998). Activation ofNMDA receptors seems to release adenosine itselfrather than a precursor (Manzoni et al., 1994; Harveyand Lacey, 1997). Dopamine D1 receptors enhance aden-osine release via an NMDA receptor-dependent increasein extracellular adenosine levels (Harvey and Lacey,1997), but dopamine depletion causes no significantchanges in the extracellular levels of striatal adenosineas measured by in vivo microdialysis (Ballarin et al.,1987). Thus, dopaminergic input may be important totransiently elevate adenosine but not so important inmaintaining a basal level of the nucleoside. Nitric oxidecan also control basal levels of endogenous adenosine invivo (Fischer et al., 1995; Delaney et al., 1998) as well asin vitro (Fallahi et al., 1996).

Another potential source of extracellular adenosine iscAMP, which can be released from neurons and con-verted by extracellular phosphodiesterases into AMPand thereafter by an ecto-5�-nucleotidase to adenosine.Functional evidence for a relevant role of this pathwayhas been obtained in the ventral tegmental area andhippocampus (Bonci and Williams, 1996; Brundege etal., 1997; Dunwiddie et al., 1997a,b). However, to pro-vide a physiologically important adenosine release, itseems that multiple cells must release cAMP over aprolonged period (Brundege et al., 1997).

Levels of adenosine in the rodent and cat brain havebeen determined by different methods including freeze-blowing (Winn et al., 1981), high-energy focused micro-wave irradiation (Delaney et al., 1998; Delaney and Gei-ger, 1998) and microdialysis (Zetterstrom et al., 1982;Porkka-Heiskanen et al., 1997) and have been estimatedto be approximately 30 to 300 nM. These levels aresufficient to cause activation of adenosine A1 and A2Areceptors.

The levels of adenosine, at least in the basal forebrain,striatum, hippocampus, and thalamus, are higher dur-ing wakefulness than sleep (Huston et al., 1996; Porkka-Heiskanen et al., 1997). The highest levels of adenosinein hippocampus were estimated during the hours beforerats entered into a sleep-like behavior, suggesting that

adenosine has sleep-promoting properties (Huston et al.,1996). Moreover, extracellular adenosine levels in-creased 2-fold in the basal forebrain of the cat after 4 hof handling to ensure prolonged wakefulness (Porkka-Heiskanen et al., 1997).

As is well known, levels of adenosine increase, up to100-fold, as a result of oxidative stress and ischemia(Rudolphi et al., 1992a; Latini et al., 1999). Excitatoryamino acid-mediated release of adenosine is certainlyinvolved; however, of greater importance is probably thefact that whenever intracellular levels of adenine nucle-otides fall as a result of excessive energy use, the intra-cellular levels of adenosine will rise dramatically (Ru-dolphi et al., 1992a). For example, following hypoxia(Zetterstrom et al., 1982), ischemia (Berne et al., 1974),or electrical stimulation (Pull and McIlwain, 1972),there is a decrease of intracellular ATP, accompanied byan accumulation of 5�-AMP and subsequently adeno-sine. The nucleoside is thereafter transported into theextracellular space via the above-mentioned transport-ers (Jonzon and Fredholm, 1985; Fredholm et al.,1994b). An elegant illustration of the capacity of thesetransporters to move adenosine from the intra- to theextracellular space was provided by loading a high con-centration of adenosine into a single hippocampal CA 1neuron and shortly thereafter, identifying in the samecell an inhibition of the excitatory postsynaptic potentialmediated by extracellular adenosine (Brundege andDunwiddie, 1996). Furthermore, when the intracellularlevel of adenosine is very high, adenosine simply diffusesout of cells. Direct release of intracellular adenine nu-cleotides, such as ATP, that is thereafter converted ex-tracellularly by ecto-ATPase and ecto-ATP-diphospho-hydrolase (ecto-apyrase) to AMP and dephosphorylatedby ecto-5�-nucleotidase to adenosine, should also be con-sidered (Rudolphi et al., 1992a; Zimmermann et al.,1998).

IV. Structure

By now all four adenosine receptors have been clonedfrom rat, mouse, and human (the structural informationis available e.g., via GPCRDB www.gpcr.org/7tm/). Inaddition, A1 receptors are cloned from dog, cow, rabbit,guinea pig, and chick; the A2A receptor from dog andguinea pig; the A2B receptor from chick; and the A3receptor from dog, sheep, rabbit, and chick. As seen fromthe dendrogram in Fig. 1, there is a close similaritybetween receptors of the same subtype, at least amongmammals. The largest variability is seen for the A3receptor for which there is almost a 30% difference atthe amino acid level between human and rat. This dif-ference is in fact larger than that between human andchick A1 receptors.

The four adenosine receptor subtypes are asparagine-linked glycoproteins and all but the A2A have sites forpalmitoylation near the carboxyl terminus (Linden,

530 FREDHOLM ET AL.

2001). Depalmitoylation of A3 (but not A1) receptorsrenders them susceptible to phosphorylation by G pro-tein-coupled receptor kinases (GRKs), which in turn re-sults in rapid phosphorylation and desensitization(Palmer and Stiles, 2000).

It has long been known that A1 and A3 receptorscouple to Gi/o and that A2A and A2B receptors couple toGs (see Section IX.). Experiments with chimeric A1/A2Areceptors indicate that structural elements in both thethird intracellular loop and the carboxyl terminus influ-ence coupling of A1 receptors to Gi, whereas elements inthe third intracellular loop but not the carboxyl termi-nus contribute to A2A receptor coupling to Gs (Tucker etal., 2000). Reconstitution experiments have revealedthat the coupling of A1 receptors is influenced by thecomposition, prenylation state (Yasuda et al., 1996) andphosphorylation state (Yasuda et al., 1998) of G protein�-subunits. A2A receptors vary in their affinity for Gsproteins containing various types of �-subunits and in-teract most avidly with G proteins containing �4 (McIn-tire et al., 2001).

V. Gene Structure

The genomic structure appears to be similar for all thehuman adenosine receptors. There is a single intron thatinterrupts the coding sequence in a region correspondingto the second intracellular loop (Ren and Stiles, 1994;Fredholm et al., 2000; Olah and Stiles, 2000). The beststudied receptor is the A1 receptor. Already when thestructure of the A1 receptor was first reported, the pres-ence of two major transcripts was noted. It was origi-nally thought that they might represent alternativesplicing, and more recent data have yielded additional

information (Ren and Stiles, 1994, 1995). Transcriptscontaining three exons, called exons 4, 5, and 6 werefound in all tissues expressing the receptor, whereastranscripts containing exons 3, 5, and 6 are in additionfound in tissues such as brain, testis, and kidney, whichexpress high levels of the receptor. There are two pro-moters, a proximal one denoted promoter A, and a distalone denoted promoter B, which are about 600 base pairsapart. Promoter B and exon 1B are part of an intronwhen promoter A is active (Ren and Stiles, 1995). Bothpromoters were suggested to have nontraditional TATAboxes.

Reporter assay studies in DDT1 MF-2 cells show that500 base pairs of promoter A contained essential ele-ments for A1 receptor expression, and mice expressingpromoter A driving the �-galactosidase reporter geneconfirmed this (Rivkees et al., 1999b). Furthermore, thispromoter contained binding sites for GATA and forNkx2.5, which factors individually drive promoter activ-ity and also act synergistically (Rivkees et al., 1999b).Promoter B has been shown to be activated by, amongother things, glucocorticoids (Ren and Stiles, 1999). It isknown that glucocorticoids can stimulate the expressionof A1 receptors in DDT1 MF-2 cells (Gerwins and Fred-holm, 1991) and in brain (Svenningsson and Fredholm,1997). The exact reason for this is unknown, since nei-ther promoter contains a canonical glucocorticoid re-sponse element (Ren and Stiles, 1999), but interactionswith e.g., SRE-2 elements and AP-1 sites may be in-volved. The magnitude of the glucocorticoid effect thatcould be shown using reporter constructs was muchhigher when promoter B acted alone than when bothpromoters were present and active (Ren and Stiles,1999). In DDT1 MF-2 cells and in brain, promoter Aappears important (Rivkees et al., 1999b).

The cloning of much of chromosome 22, where the A2A

receptor is located (MacCollin et al., 1994), suggested atwo exon structure (Fredholm et al., 2000), which issimilar to that reported for the rat A2A receptor (Chu etal., 1996; Peterfreund et al., 1996). By comparing thehuman sequence data with data from rodents, putativeregulatory elements were identified, including AP-1, NF1 and AP-4 elements (Fredholm et al., 2000). The A2A

receptor shows one hybridizing transcript in most tis-sues examined (Maenhaut et al., 1990; Stehle et al.,1992; Peterfreund et al., 1996). However, examination ofRNA isolated from PC12 cells suggested two differentstart sites (Chu et al., 1996). The expression of A2A

receptor can be stimulated by protein kinase C (Peter-freund et al., 1997) and hypoxia (Kobayashi et al., 1998).We do not know the transcription factors involved ineither case. It should also be mentioned that the humanadenosine A2A receptor is polymorphic. In particular, a(silent) T1083C mutation occurs in various populations,more frequently in caucasians than in Asians (Deckertet al., 1996; Le et al., 1996; Soma et al., 1998).

FIG. 1. Dendrogram showing sequence similarity between clonedadenosine receptors. The figure is slightly redrawn from that available athttp://www.gpcr.org/7tm/seq/007_001/001_007_001.TREE.html. This phylo-genetic tree was automatically calculated by WHAT IF based on a neighbor-joining algorithm.


Analysis of the A2B receptor gene, localized on chro-mosome 17 in man, reveals a similar overall structure asfor the other adenosine receptors. The rat A2B receptorshows two hybridizing transcripts of 1.8 and 2.2 kb,where the latter is the dominant one (Stehle et al.,1992). This could, in analogy with the above, suggest thepresence of multiple promoters, but so far this has notbeen studied to our knowledge.

The mouse A3 receptor appears to have two exons withcoding sequences of 354 and 1135 base pairs separatedby an intron of about 2.3 kb (Zhao et al., 1999). Severalputative transcription factor-binding sites could be de-tected in the mouse gene (Zhao et al., 1999), but surpris-ingly, few of these are matched by similar elements inthe human gene. This could mean that the truly impor-tant sites have not been identified, or else that theexpression of the receptor is regulated very differently inthe two species. The fact that the distribution of A3receptors in humans and rodents is very different mightindicate the latter. The human A3 receptor shows twotranscripts: the most abundant is approximately 2 kb insize, and the much less abundant one is about 5 kb(Atkinson et al., 1997). There are several possible expla-nations for this, one of which being a similarity with theA1 receptor gene.

VI. Binding Sites As Revealed by Site-DirectedMutagenesis

Adenosine receptors, like the other G protein-coupledreceptors (GPCR), are integral membrane proteins.Such macromolecules are not easily amenable to crys-tallization and, hence, to precise structure elucidationthrough X-ray diffraction. However, substantialprogress has been made over the last decade or so inunraveling the three-dimensional architecture of tworelated membrane-bound proteins, i.e., bacteriorhodop-sin and (mammalian) rhodopsin. The pivotal suggestion

that the GPCR family bears structural homology tothese two proteins has been an impetus to our currentunderstanding of receptor structure. Bacteriorhodopsin,a proton pump present in the cell wall of Halobacteriumhalobium, and rhodopsin, itself a G protein-coupled re-ceptor, are of similar size and share many characteris-tics between themselves and with other mammalian Gprotein-coupled receptors. They both have the typicalseven-transmembrane �-helical architecture and bindretinal, their endogenous ligand, in the cavity formed bythe barrel-like arrangement of the seven transmem-brane domains. On the other hand there is little se-quence (i.e., amino acid) homology between the two pro-teins and an almost total lack of homology betweenbacteriorhodopsin and G protein-coupled receptors. Hi-bert and coworkers were the first to realize and analyzein depth the opportunities and pitfalls of using theatomic coordinates of bacteriorhodopsin, at that timeavailable at low resolution only (Henderson et al., 1990),and later, of rhodopsin, to construct putative receptormodels (Hibert et al., 1991; Hoflack et al., 1994). Insubsequent years ever greater resolution and accuracywere achieved (Kimura et al., 1997; Unger et al., 1997),eventually resulting in the elucidation of the structuresof bacteriorhodopsin (Fig. 2A) and rhodopsin (Fig. 2B) at1.55 and 2.8 Å, respectively (Luecke et al., 1999; Palc-zewski et al., 2000).

It is obvious that so-called homology modeling, i.e., theconstruction of a three-dimensional model of a givenprotein (e.g., one of the adenosine receptor subtypes) onthe basis of an experimentally determined structure ofanother related protein (e.g., bacteriorhodopsin or rho-dopsin) can only generate highly speculative models.This is particularly true when it comes to apparentdifferences between the macromolecules under study,for instance in their ligand binding sites. Nevertheless, anumber of receptor models have been developed on the

FIG. 2. Protein backbone representations of the structures of bacteriorhodopsin (A) and rhodopsin (B) as retrieved from the Brookhaven ProteinData Bank (1C3W and 1F88, respectively).

532 FREDHOLM ET AL.

basis of either bacteriorhodopsin or rhodopsin (at vari-ous degrees of resolution). Thus, Baldwin combinedstructural information on rhodopsin with a sequenceanalysis of other GPCRs to suggest a probable arrange-ment (including “borders”) of the seven �-helices (Bald-win et al., 1997). This template structure was used toprovide models for all G protein-coupled receptors in anautomated fashion, which can be easily retrieved fromthe internet (for information on G protein-coupled recep-tors, including these three-dimensional models, see theGPCR Database http://www.gpcr.org/7tm/). Althoughmet with skepticism, such receptor models have beenuseful in clarifying the putative molecular basis of re-ceptor-ligand recognition, in particular when combinedwith and adjusted to available pharmacological andstructure-activity relationship data.

In the adenosine receptor field, the first receptormodel targeted the adenosine A1 receptor (IJzerman etal., 1992) based on the sequence of the canine orphanreceptor RDC7, later identified as an A1 receptor (Libertet al., 1989, 1991), and the low resolution structure ofbacteriorhodopsin (Henderson et al., 1990). It was foundthat the pore formed by the seven amphipathic �-heliceswas characterized by a rather distinct partition betweenhydrophobic and hydrophilic regions. Chemical modifi-cation of histidine residues in the receptor, of which twoare present in the transmembrane domains, one in helixVI and one in helix VII, strongly affects ligand binding.This provided the basis for docking the potent and A1-selective agonist N6-cyclopentyladenosine into this cav-ity. A similar model, again based on the bacteriorhodop-sin structure and the two histidine residues, wasdeveloped for the rat adenosine A2A receptor (IJzermanet al., 1994).

Later, mutation studies indicated these and otheramino acid residues as being important for either ago-nist or antagonist binding or both (see next four para-graphs). This led to further but similar models for in-stance for the human A2A receptor, based on the lowresolution structure of rhodopsin (Kim et al., 1995). Ascan be inferred from Fig. 2, A and B, the two rhodopsinproteins are similar but certainly not identical in theirtransmembrane organization (at the time of the firstreceptor models, there was no structural information onthe extracellular and intracellular domains). Studies ofan increasing number of point mutations (often con-ceived and selected from the receptor models) led tofurther insight in the ligand binding site, giving rise tosome refinement of the existing models. In Fig. 3 asnapshot of the most recently published adenosine A1receptor model highlights the six residues in helices IIIand VII probably involved in agonist binding (Rivkees etal., 1999a). With the recent structure elucidation of rho-dopsin (Fig. 2B), earlier results that were somewhatanomalous can be rationalized. For instance, Jacobsonand coworkers identified glutamate residues in the sec-ond extracellular loop as being important for either di-

rect or indirect ligand recognition (Kim et al., 1996). Inrhodopsin, part of this domain folds deeply into thecenter of the macromolecule, the site where ligand bind-ing in both rhodopsin and adenosine receptors is thoughtto take place. This arrangement causes the second ex-tracellular loop to be in extensive contact with both theextracellular regions and the ligand binding site. Theproteolytic analysis of A1 receptors photoaffinity-labeledwith a xanthine antagonist indicates that the site ofalkylation is in TM3 (Kennedy et al., 1996).

Extensive mutagenesis, consisting of single aminoacid replacement (typically Ala scanning), has been car-ried out for both A1 and A2A receptors (Tables 2 and 3),and to a lesser extent for A2B receptors (Table 4). Themost essential interactions required for recognition ofagonist and/or antagonist occur in TMs 3, 5, 6, and 7.Two His residues (6.52 and 7.43) are conserved amongmost of the adenosine receptor subtypes, with the excep-tion of the A3 receptor, which lacks His at 6.52. TheseHis residues are important for ligand recognition (Olahet al., 1992; Kim et al., 1995; Gao et al., 2000). Mutationof His at 6.52 to Leu in the A1 receptor selectively weak-ened binding of an antagonist, whereas at 7.43 the samemutation impaired both agonist and antagonist binding(Olah et al., 1992). When either of these residues in theA2A receptor was mutated to Ala, there was a dramaticloss of affinity for both agonist and antagonist (Kim etal., 1995). Some substitutions by amino acids havingaromatic and other side chains partially restored func-tion of the binding site. At the A2A receptor, substitutionat 7.43 with Tyr selectively reduced agonist affinity (Gaoet al., 2000). The hydrophilic residue at 7.42 (Thr in A1and Ser in A2A receptors; also see Fig. 3) when mutatedto Ala caused a significant loss of affinity for agonistswhile having little effect on antagonist binding(Townsend-Nicholson and Schofield, 1994; Kim et al.,1995). The hydrophobic residue at 7.35 in the A1 recep-tor (Ile in bovine and Met in canine) appeared to corre-

FIG. 3. Computer modeling of CPA (yellow), human A1 adenosinereceptor interactions. Only helices III (left) and VII (right) are shown(both in green) with relevant residues (half-bond colors).


late with the species-related differences in agonist phar-macology (Tucker et al., 1994). Mutation of an Asn at7.36 of the A2B receptor to Tyr, the homologous residueat the other subtypes, selectively enhanced the affinityof 2-substituted agonists (Beukers et al., 2000).

TM3 in the A2A receptor contains a sequence of fourhydrophilic amino acids, of which the first two (Thr at3.36 and Gln at 3.37) when mutated had major effects onligand affinity (Jiang et al., 1996). An enhancement ofaffinity for N6-substituted adenosine derivatives (140-fold for IB-MECA) in the Gln to Ala mutant appeared tocorrelate inversely with size of the amino acid sidechain. At the A1 receptor, mutation of the identical res-idues at 3.36 and 3.37 (Fig. 2) to Ala impaired agonist

binding and caused a structure-dependent reduction ofantagonist affinity (Rivkees et al., 1999a).

Modulatory residues have also been found in TM1,including a Glu residue (1.39) in both A1 and A2A recep-tors, which affects agonist binding selectively (Barb-haiya et al., 1996; IJzerman et al., 1996). A Gly to Thrmutation in TM1 (1.37) of the A1 receptor increasesagonist affinity (Rivkees et al., 1999a). At the A2B recep-tor, mutation at the same position had no functionalconsequences (Beukers et al., 2000). A conserved Aspresidue in TM2 (2.50) is the site of binding of Na�, whichregulates agonist affinity (Barbhaiya et al., 1996). Nomutations of TM4 in any of the adenosine receptors haveyet been found to affect ligand binding.

TABLE 2Mutational analysis of the adenosine A1 with respect to ligand binding

Mutationa Species Helix/positionb Effect on Ligand Binding and Signal Transduction Reference

G14T H 1.37 Increased agonist affinity Rivkees et al., 1999aE16A/Q H 1.39 Agonist affinity reduced 4- to 40-fold; little change in

antagonist affinityBarbhaiya et al., 1996

P25L H 1.48 Modest reduction of agonist affinity Rivkees et al., 1999aI31C H 1.54 No changes in radioligand binding Rivkees et al., 1999aC46A/S H 2.41 No changes in radioligand binding Scholl and Wells, 2000S50A H 2.45 No changes in radioligand binding Barbhaiya et al., 1996D55A H 2.50 Increase in agonist affinity with no change in antagonist

affinity; disrupted regulation of agonist binding by sodiumions

Barbhaiya et al., 1996

L65F H 2.60 No changes in radioligand binding Rivkees et al., 1999aC80A/S H 3.25 No detectable radioligand binding Scholl and Wells, 2000M82F H 3.27 No changes in radioligand binding Rivkees et al., 1999aC85A H 3.30 No changes in radioligand binding Scholl and Wells, 2000C85S Agonist affinity reduced 4- to 13-fold; no change in

antagonist affinityScholl and Wells, 2000

P86F H 3.31 Substantial reduction of agonist binding Rivkees et al., 1999aV87A H 3.32 No change in ligand affinity Rivkees et al., 1999aL88A H 3.33 Substantial reduction of agonist binding, but also of N-0840

antagonist bindingRivkees et al., 1999a

T91A H 3.36 Substantial reduction of agonist binding, but also of N-0840antagonist binding

Rivkees et al., 1999a

Q92A H 3.37 Substantial reduction of agonist binding, but also of N-0840antagonist binding

Rivkees et al., 1999a

S93A H 3.38 No changes in radioligand binding Barbhaiya et al., 1996S94A H 3.39 No detectable agonist or antagonist binding Barbhaiya et al., 1996S94T Minor changes in ligand binding Barbhaiya et al., 1996A125K H 4.43 No changes in radioligand binding Rivkees et al., 1999aC131A/S H 4.49 No changes in radioligand binding Scholl and Wells, 2000S135A H 4.53 No changes in radioligand binding Barbhaiya et al., 1996T141A H 4.59 No changes in radioligand binding Barbhaiya et al., 1996F144L H 4.62 No changes in radioligand binding Rivkees et al., 1999aC169A/S H No detectable radioligand binding Scholl and Wells, 2000H251L B 6.52 Antagonist affinity reduced 4-fold; no change in agonist

affinityOlah et al., 1992

C255A/S H 6.56 No changes in radioligand binding Scholl and Wells, 2000C260A/S H No changes in radioligand binding Scholl and Wells, 2000C263A/S H No changes in radioligand binding Scholl and Wells, 2000I270M M270I B, C 7.35 Amino acid in position 270 contributes to canine/bovine A1

AR binding selectivityTucker et al., 1994

T277A H 7.42 400-fold decrease in affinity of NECA with modest changesin affinity for R-PIA and S-PIA (intact cells); substantialdecrease in affinity of all agonists (membranes); no changein antagonist affinity

Townsend-Nicholsonand Schofield, 1994;Dalpiaz et al., 1998

T277S Modest decrease in agonist affinity; no change in antagonistaffinity; nature of residue in position 277 also involved incanine/bovine A1 AR binding specificity

Tucker et al., 1994;Townsend-Nicholsonand Schofield, 1994

H278L B 7.43 Negligible agonist and antagonist binding Olah et al., 1992C309A/S H No changes in radioligand binding Scholl and Wells, 2000

The results were obtained from site-directed mutagenesis studies of the A1 AR (species; H, human; B, bovine; C, canine).a Amino acids are represented in single-letter code with position number shown. The first amino acid is that of the wild-type receptor, with the second residue being that

used for substitution.b Position on helix, using notation of van Rhee and Jacobson (1996).

534 FREDHOLM ET AL.

Negatively charged residues in the second extracellu-lar loop of the A2A receptor have been found to be re-quired for binding of both agonist and antagonist (Kimet al., 1996). Among nine native Cys residues of thehuman A1 receptor, only a single pair, Cys80 andCys169, were found to be essential for ligand binding(Scholl and Wells, 2000). This pair corresponds to Cysresidues conserved among rhodopsin-like GPCRs, whichform a disulfide bridge required for the structural integ-rity of the receptor.

VII. Distribution

It is important to study the distribution of receptors,because this will tell us where agonists and antagonists

given to the intact organism can act. Furthermore, ingeneral, the higher the number of receptors the morepotent and/or efficacious will be the agonist. Thus, therather low levels of endogenous adenosine present underbasal physiological conditions have the potential of ac-tivating receptors where they are abundant, but notwhere they are sparse (Kenakin, 1993, 1995; Svennings-son et al., 1999c; Kull et al., 2000b; Fredholm et al.,2001).

There is much information on the distribution of theA1 and A2A receptors because good pharmacologicaltools including radioligands (see below) are available.There are also several studies that have used antibodiesto localize adenosine A1 receptors in brain (Swanson et

TABLE 3Mutational analysis of the human A2A AR with respect to ligand binding

Mutationa Helix/Positionb Effect on Ligand Binding and Signal Transduction Reference

E13Q 1.39 Slight reduction of agonist, but not antagonist, affinity IJzerman et al., 1996T88A/S/R 3.36 Substantial decrease in agonist, but not antagonist, affinity Jiang et al., 1996Q89A 3.37 Slight increase in agonist and antagonist affinity Jiang et al., 1996Q89N/S/L Marginal changes in ligand bindingQ89H/R Antagonist binding affectedS90A 3.38 Marginal changes in ligand binding Jiang et al., 1996S91A 3.39 Marginal changes in ligand binding Jiang et al., 1996E151A/Q/D Loss of agonist and antagonist radioligand binding Jiang et al., 1996E169A �1000-fold decrease in agonist potency Kim et al., 1996E169A Gain in N6-substituted agonist affinity Kim et al., 1996D170K No change in (radio)ligand binding Kim et al., 1996P173RF180A 5.41 Minor changes in ligand binding Kim et al., 1995N181S 5.42 Modest reduction of agonist binding Kim et al., 1995F182A 5.43 Loss of agonist and antagonist radioligand binding Kim et al., 1995F182Y Modest reduction of agonist binding Kim et al., 1995F182W Modest reduction of agonist binding Kim et al., 1995H250A 6.52 Loss of agonist and antagonist radioligand binding; no agonist activity

in functional assaysKim et al., 1995

H250F/Y Modest reduction of agonist binding; no effect on antagonist binding Kim et al., 1995N253A 6.55 Loss of agonist and antagonist radioligand binding Kim et al., 1995C254A 6.56 Minor changes in ligand binding Kim et al., 1995F257A 6.59 Loss of agonist and antagonist radioligand binding Kim et al., 1995C262G 6.64 No change in radioligand binding Kim et al., 1996I274A 7.39 Loss of agonist and antagonist radioligand binding; �30-fold decrease

in agonist potencyKim et al., 1995

S277A 7.42 Substantial decrease in agonist affinity and potency, but antagonistradioligand binding not altered

Kim et al., 1995

S277T/C/N Marginal changes in ligand binding Jiang et al., 1996H278A 7.43 Loss of agonist and antagonist radioligand binding; �300-fold decrease

in agonist potencyKim et al., 1995

H278Y Modest reduction of agonist binding; no effect on antagonist binding Gao et al., 2000S281A 7.46 Loss of agonist and antagonist radioligand binding; no agonist activity

in functional assaysKim et al., 1995

S281T Enhanced affinity for most agonists Kim et al., 1995S281N Marginal changes in ligand binding Gao et al., 2000a Amino acids are represented in single-letter code with position number shown. The first amino acid is that of the wild-type receptor, with the second and further residues

those used for substitution.b Position on helix, using notation of van Rhee and Jacobson (1996).

TABLE 4Mutational analysis of the human A2B AR with respect to ligand binding

Mutationa Helix/Positionb Effect on Ligand Binding and Signal Transduction Reference

V11I 1.36 No changes in (radio)ligand binding Beukers et al., 2000A12T 1.37 No changes in (radio)ligand binding Beukers et al., 2000L58V 2.55 No changes in (radio)ligand binding Beukers et al., 2000F59L 2.56 No specific radioligand binding and no cAMP production Beukers et al., 2000N273Y 7.36 Up to 60-fold increase in affinity of 2-substituted adenosines Beukers et al., 2000

a Amino acids are represented in single-letter code with position number shown. The first amino acid is that of the wild-type receptor, and the second is the one used forsubstitution, based on the corresponding amino acid in the human A2A receptor.

b Position on helix, using notation of van Rhee and Jacobson (1996).


al., 1995; Saura et al., 1998; Middlekauff et al., 1998)and A2A receptors in striatum (Rosin et al., 1998; Het-tinger et al., 2001), carotid body (Gauda et al., 2000), andT cells (Koshiba et al., 1999). In the case of the A2B andA3 receptors, the data are less impressive. Here onetends to rely on data on the expression of the correspond-ing mRNA. Some of this information is summarized inTable 5. The results presented there clearly show thatthere is much left to examine regarding the distributionof especially A2B and A3 receptors. Furthermore, it islikely that a better understanding of the transcriptionalregulation (see above) will be of considerable help inunderstanding the spatio-temporal aspects of adenosinereceptor distribution.

Receptor protein and the corresponding message areoften colocalized but there are important differences.For example, in several regions of the central nervoussystem, receptor binding and expression of transcript donot exactly match (Johansson et al., 1993a), and the twoare differently regulated by e.g., long term antagonisttreatment (Johansson et al., 1993a) and during develop-ment (Åden et al., 2000, 2001). Much of the differentialdistribution can probably be explained by the fact that asubstantial number of adenosine A1 receptors arepresent at nerve terminals. A similar explanation prob-ably underlies the observations that A2A receptors arepresent in globus pallidus, despite the fact that A2Areceptor mRNA cannot be detected there (Svenningssonet al., 1997, 1999c). These receptors are probably locatedat the terminals of the striatopallidal GABAergic neu-rons (Rosin et al., 1998; Svenningsson et al., 1999b;Linden, 2001).

Besides regulation at the level of gene transcription,targeting of the receptor protein to different locationswithin the cell is crucial. This important aspect of recep-tor distribution is just starting to be explored in the caseof adenosine receptors. Recently it was found that inMDCK cells (a canine kidney cell line) the adenosine A1receptor is targeted to the apical surface, whereas the�-adrenoceptor, which is also Gi coupled, is directed tothe basolateral surface (Saunders et al., 1996). The dis-tribution is highly dependent on the third intracellularloop and/or the carboxyl-terminal segment of the recep-tor as judged by the distribution of receptor chimeras(Saunders et al., 1998). Interestingly, the apical distri-bution of adenosine A1 receptors was disrupted byagents that interfere with microtubules, whereas thebasolateral distribution of � adrenergic receptors wasnot (Saunders and Limbird, 1997). Thus, G protein-coupled receptors appear to use several different target-ing mechanisms. This is also borne out by the fact thatdistribution of these receptors in the kidney epithelialcell line does not predict distribution in neurons(Wozniak and Limbird, 1998).

VIII. Classification of Adenosine Receptors UsingPharmacological Tools

Since adenosine receptors have been studied for a longtime there are several useful pharmacological tools (Ta-ble 6). The structures of some typical drugs used inreceptor classification are shown in Fig. 4. Data on theirbinding to human and rat receptors are presented inTables 7 and 8. These tables contain information on

TABLE 5Summary of the distribution of adenosine receptors

Data where there is correspondence between mRNA distribution and receptor distribution are given in bold. Data based mainly or exclusively ondistribution of mRNA are given in ordinary letters. Ambiguous data (including major species differences) are given in italics.

A1 Receptor A2A Receptor A2B Receptor A3 Receptor

High expression High expression High expression High expressionBrain (cortex, cerebellum,hippocampus). Dorsalhorn of spinal cord. Eye,adrenal gland, atria

Spleen, thymus,leukocytes (bothlymphocytes andgranulocytes), bloodplatelets.StriatopallidalGABAergic neurons(in caudate-putamen,nucleus accumbens,tuberculumolfactorium), olfactorybulb

Cecum, colon, bladder Testis (rat), mast cells (rat)

Intermediate levels Intermediate levels Intermediate levels Intermediate levelsOther brain regions.Skeletal muscle, liver,kidney, adipose tissue,salivary glands,esophagus, colon, antrum,testis

Heart, lung, bloodvessels

Lung, blood vessels, eye, medianeminence, mast cells

Cerebellum (human?), hippocampus(human?), lung, spleen (sheep),pineal

Low levels Low levels Low levels Low levelsLung (but probably higherin bronchi), pancreas

Other brain regions Adipose tissue, adrenal gland,brain, kidney, liver, ovary,pituitary gland

Thyroid, most of brain, adrenalgland, spleen (human), liver,kidney, heart, intestine, testis(human)

536 FREDHOLM ET AL.

some of the most widely used compounds, but a largenumber of other compounds have also been examinedover the years using more or less selective functionalassay systems (see Section XI.). Here we will just com-ment on a few points of more general interest not con-tained in the tables.

Ideally, agonists and antagonists should differ in po-tency by at least two orders of magnitude at differentreceptors to be really useful in receptor classification. Itis apparent from Tables 7 and 8 that this is rarely thecase for any of the compounds often used in classifyingadenosine receptors. Nevertheless, with a judicious useof agonists and antagonists at A1, A2A, and A3 receptorsin in vitro experiments, strong conclusions can bedrawn. The situation is less fortunate in vivo as thepharmacokinetics of these compounds have not beenstudied extensively. It has been found, however, that theA1-selective agonist CPA has a terminal half-life in con-scious rats of 6 min in blood (Mathot et al., 1993),whereas the half-life of the A3-selective agonist Cl-IB-MECA is significantly longer, i.e., 62 min (Van Schaicket al., 1996). The relatively short half-life of CPA may bedue to its substantial uptake into erythrocytes (Pavanand IJzerman, 1998).

In the case of human, rat, and mouse A1 receptors thefull agonist CCPA (and to a somewhat lesser extentCPA) and the antagonist DPCPX are quite useful. Aminor problem with DPCPX is that it also interacts withappreciable affinity with A2B receptors. This does not

appear to be a major problem, however, since binding ofDPCPX is virtually eliminated in animals lacking the A1receptor (Johansson et al., 2001). For the adenosine A1receptor, partial agonists are available as well. Carefulchemical manipulation of the CPA molecule yielded suchcompounds, for instance by substitution at the C8-posi-tion (Roelen et al., 1996) or by modification of the ribose5�-substituent (van der Wenden et al., 1998). These com-pounds showed tissue selectivity in vivo, exploiting dif-ferences in receptor density, and in the efficiency ofreceptor coupling to further signal transduction (Mathotet al., 1995; van Schaick et al., 1998).

Another interesting class of compounds acting onadenosine A1 receptors are the so-called allosteric en-hancers. The prototype here is PD81723 (Bruns andFergus, 1990), which has been shown by various re-search groups to (allosterically) increase agonist bindingand effect (e.g., Linden, 1997). In recent years, analogsof PD81723 have been synthesized that show similareffects (van der Klein et al., 1999; Baraldi et al., 2000b).

NECA was long considered to be a selective adenosineA2 receptor agonist, but as seen from the tables this viewcan no longer be upheld. Based on evidence that 2-sub-stitution of NECA increased selectivity, CGS 21680 wasdeveloped as an A2A receptor-selective agonist (Hutchi-son et al., 1989). However, in humans it is less potentand less selective than in rats (Kull et al., 1999). Indeed,there are clear-cut differences in the order of potency ofagonists, but not antagonists, between human and rat

TABLE 6Pharmacological tools used for classification of adenosine receptors

Abbreviations Chemical Names

AB-MECA 4-aminobenzyl-5�-N-methylcarboxamidoadenosineATL146e 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-

cyclohexanecarboxylic acid methyl esterBW-A1433 1,3-dipropyl-8-(4-acrylate)phenylxanthineCGS 15943 5-amino-9-chloro-2-(2-furyl)-[1,2,4]-triazolo[1,5-c]quinazolineCGS 21680 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5�-N-ethylcarboxamidoadenosineCP 66713 4-amino-8-chloro-1-phenyl-[1,2,4]-triazolo-[4,3-a] quinoxalineCPA N6-cyclopentyladenosineCPX 8-cyclopentyl-1,3-dipropylxanthine (see also DPCPX)CSC 8-(3-chlorostyryl)caffeineCV-1808 2-phenylaminoadenosineDPCPX 1,3-dipropyl-8-cyclopentylxanthineHENECA 2-hex-1-ynyl-5�-N-ethylcarboxamidoadenosineI-ABA N6-(4-amino-3-iodobenzyl)adenosineI-ABOPX 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propylxanthineIB-MECA N6-(3-iodobenzyl)adenosine-5�-N-methyluronamideKF 17837 1,3-dipropyl-8-(3,4-dimethoxystyryl)-7-methylxanthineKW 6002 (E)-8-[2-(3,4-dimethoxyphenyl)ethenyl]-1,3-diethyl-3,7-dihydro-7-methyl-1h-purine-2,6-dioneMRE 3008-F20 5-[[(4-methoxyphenyl)amino]carbonyl]amino-8-ethyl-2-(2-furyl)-pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidineMRS1191 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(�)-dihydropyridine-3,5-dicarboxylateMRS1220 9-chloro-2-(2-furanyl)-5-[(phenylacetyl)amino][1,2,4]-triazolo[1,5-c]quinazolineMRS1523 2,3-diethyl-4,5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylateMRS1754 N-(4-cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]acetamideNECA 5�-N-ethyl-carboxamidoadenosinePAPA-APEC 2-(4-[2-[(4-aminophenyl)methylcarbonyl]ethyl]phenyl)ethylamino-5�-N-ethylcarboxamidoadenosinePD 81723 (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)-phenyl]-methanoneR-PIA R-N6-(phenylisopropyl)-adenosineSCH 58261 5-amino-2-(2-furyl)-7-phenylethyl-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidineS-PIA S-N6-(phenylisopropyl)-adenosineVUF-8504 4-methoxy-N-[2-(2-pyridinyl)quinazolin-4-yl]benzamideXAC xanthine amine congener; 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthineZM241385 4-(2-[7-amino-2-[2-furyl]-[1,2,4]triazolo[2,3-a]{1,3,5}triazin-5-yl-amino]ethyl)phenol


A2A receptors (Kull et al., 1999). There is an additionalproblem with CGS 21680 as a tool; it also binds to sitesunrelated to A2A receptors (Johansson et al., 1993b;Johansson and Fredholm, 1995; Cunha et al., 1996;

Lindstrom et al., 1996). This means that at least inorgans or cells with few A2A receptors, effects of CGS21680 must be viewed with skepticism. ATL146e wasrecently developed as a new A2A agonist that is over 50

FIG. 4. Structures of reference compounds used to classify adenosine receptors. Panel A, structure of some non-selective and A1 receptor-selectiveadenosine analogs; panel B, structure of adenosine analogs used to classify A2A and A3 receptors; panel C, structure of selected adenosine receptorantagonists.

538 FREDHOLM ET AL.

times more potent than CGS 21680 at the human recep-tor (Rieger et al., 2001). It has strong in vivo effects one.g., reperfusion injury in the rabbit lung (Ross et al.,1999) and the rat kidney (Okusa et al., 1999), and re-duces expression of adhesion molecules on the reper-fused vascular endothelium (Okusa et al., 2000). Thereare several useful A2A receptor antagonists. The mostselective so far is SCH 58261. The structurally relatedZM 241385 is more readily available (Poucher et al.,1995), but shows appreciable affinity to A2B receptors(Ongini et al., 1999).

The adenosine A2B receptor has low affinity for mostagonists. For the other receptors, agonists with potencyin the low nanomolar range are available, but in the case

of A2B receptors the most potent agonists have affinitiesonly marginally below 1 �M. Furthermore, selectivity isnegligible. The situation is somewhat more favorable inthe case of antagonists, where some potent and rela-tively selective antagonists have been found (Kim et al.,2000).

The most recently discovered adenosine receptor—theA3 receptor—is notably insensitive to several xanthines.Hence, most A3 antagonists have a nonxanthine struc-ture, including dihydropyridines, pyridines, and fla-vonoids (Baraldi et al., 2000a). Isoquinoline and quina-zoline derivatives constitute another class of highlyselective human A3 receptor antagonists. VUF8504(4-methoxy-N-[2-(2-pyridinyl)quinazolin-4-yl]benz-

TABLE 7Binding affinity of agonists and antagonists at human adenosine receptor subtypes (Ki values with 95% confidence intervals or � S.E.M. in

parentheses)

A1 A2A A2B A3 Reference

Agonists with unmodified riboseR-PIA 2.0 (1–4.2) 860 (480–1,540) 16 (9.3–29) —a

33,700 (�6,800) —c

3,800 (�1,700) —e

S-PIA 75 (42–134) 7,800 (7,000–8,640) 45 (33–61) —a

62,800 (�21,600) —c

CPA 2.3 (1.5–3.4) 790 (470–1,360) 43 (30–61) —a

34,400 (�11,100) —c

21,000 (�4,300) —e

CCPA 0.8 (0.55–1.25) 2,300 (2,000–2,700) 42 (32–56) —a

40,100 (�16,400) —c

2-Cl-adenosine 1.39 (1.28–1.51) 180 (150–220) 19 (14–28) —i

Agonists with 5�-modified riboseNECA 14 (6.4–29) 20* (12–35) 6.2* (3.5–11) —a

330 (�60) —c

360 (�120) —e

CGS 21680 290 (230–360) 27 (12–59) 67 (50–90) —a

361,000 (�21,000) —c

HENECA 60 (50–72) 6.4 (3.8–11) 2.4 (2.0–2.9) —b

PENECA 560 (480–650) 620 (300–1,300) 6.2 (5.1–7.5) —b

PHPNECA 2.7 (1.7–7.1) 3.1 (2.4–3.9) 0.42 (0.17–1.0) —b

AB-MECA 1,500 (1,300–1,800) 3,600 (2,700–4,700) 22 (20–24) —a

IB-MECA 3.7 (1.8–7.7) 2,500 (1,900–3,400) 1.2 (0.96–1.5) —a

54,000 (�5,600) —c

IAB-MECA 8.5 (4.8–15) 470 (300–740) 0.64 (0.58–0.70) —a

Cl-IB-MECA 115 (114–116) 2,100 (1,700–2,500) 11 (9.4–13) —b

Antagonists: xanthinesDPCPX 3.9* (3.5–4.2) 129 (35–260) 4,000 (2,600–6,000) —a

50 (�3.7) —c

51 (�6.1) —e

XAC 29 (24–35) 1 (0.58–1.7) 92 (64–130) —a

7.3 (�2.8) —c

12 (�4.6) —e

MRS1754 400 (�190) 500 (�11) 2.0 (�0.31) 570 (�180) —f

MSX-2 2,500 (1,700–3,600) 5.0 (�0.6) �10,000 —h

Theophylline 6,800 (4,100–11,000) 1,700 (1,000–2,900) 86,000 (74,000–101,000) —a

Antagonists: non-xanthinesCGS 15943 3.5 (1.7–7.3) 4.2 (2.6–6.6) 51 (43–60) —a

16 (�3.6) —e

SCH 58261 290 (210–410) 0.6 (0.5–0.7) �10,000 —d

ZM 241385 260 (190–390) 0.8 (0.7–1.0) �10,000 —d

32 (�6) —c

MRE 3008F20 1,100 (�1,100) 140 (�15) 2,100 (�300) 0.29 (�0.03) —g

* Values are from saturation experiments.a Klotz et al., 1998, transfected CHO cells; radioligands [3H]CCPA (A1), [3H]NECA (A2A and A3).b Klotz et al., 1999, transfected CHO cells; radioligands [3H]CCPA (A1), [3H]NECA (A2A and A3).c Linden et al., 1999, transfected HEK 293 cells; radioligand 125I-ABOPX.d Ongini et al., 1999, transfected CHO cells (A1 and A2A) or HEK 293 cells (A2A and A3); radioligands [3H]DPCPX (A1), [3H]SCH 58261 (A2A), 125I-AB-MECA (A3).e Ji and Jacobson, 1999, transfected HEK 293 cells; radioligand [3H]ZM 241685.f Kim et al., 2000, transfected HEK 293 cells; radioligands 125I-ABA (A1), 125I-ZM 241685 (A2A), 125I-ABOPX (A2B), 125I-AB-MECA (A3).g Varani et al., 2000, transfected CHO cells; radioligands [3H]DPCPX (A1 and A2B), [3H]SCH 58261 (A2A), 125I-AB-MECA (A3).h Sauer et al., 2000, transfected CHO cells; radioligand [3H]CHA (A1), [3H]CGS 21680 (A2A), [3H]NECA (A3).i K.-N. Klotz and S. Kachler, unpublished, transfected CHO cells; radioligand [3H]NECA.


TABLE 8Binding affinity of agonists and antagonists at rat adenosine receptor subtypes (Ki values with 95% confidence intervals or �S.E.M. in parentheses)

A1 A2A A3 Reference

Agonists with unmodified riboseR-PIA 1.2 (�0.16) 124 (�9) —a

1.3 (1.1–1.6) 730 (690–770) —b

0.51 —c

220 (�43) —d

160 (�52) —e

S-PIA 49 (�2.4) 1,800 (�380) —a

29 —f

920 (�310) —e

CPA 0.59 (�0.02) 460 (�15) —a

0.8 (0.6–1.0) 2,000 (1,400–2,900) —b

240 (�36) —e

CCPA 0.4 (0.2–0.7) 3,900 (2,500–4,700) —b

0.23 —c

240 (�71) —e

ADAC 0.85 (�0.35) —w

2-Cl-adenosine 9.3 (�0.58) 63 (�7.5) —a

1,900 (�900) —e

DPMA 140 (�23) 4.4 (�0.2) —u

3,600 (�1,700) —e

(S)-ENBA 0.3 (�0.02) 1,400 (�150) —v

Agonists with 5�-modified riboseNECA 6.3 (�0.52) 10 (�0.5) —a

11 (7.0–17) 22 (20–25) —f

3.7 —c

260 (�36) —d

CGS 21680 3100 (�470) 22 (�4.3) —g

580 (�32) —e

HENECA 136 (95–195) 3.8 (1.5–9.9) —f

PENECA 698 (611–798) 120 (112–128) —h

PHPNECA 2.5 (2.2–2.9) 0.9 (0.7–1.3) —i

AB-MECA 430 (�45) 1,600 (�180) 14 (�3) —j

IB-MECA 54 (�5) 56 (�8) 1.1 (�0.3) —j

IAB-MECA 18 (�5) 200 (�84) 1.3 (�0.18) —j

Cl-IB-MECA 820 (�570) 470 (�370) 0.33 (�0.08) —k

Antagonists: xanthinesDPCPX 0.3 340 —l

0.18* (0.16–0.20) —l

�10,000 —e

XAC 2.8 (1.9–4.2) —m

3.5 24 —l

4 50 —n

�100,000 —o

MRS 1754 17 (�3.6) 610 (�290) —o

MSX-2 900 (�10) 8 (�3) —p

Theophylline 11,000 32,000 —b

�100,000 —o

Antagonists: non-xanthinesCGS 15943 21 (�3.5) 3.3 (�1.8) —q

22 3 —n

6.4 (6.2–6.6) 1.2 (1.1–1.3) —r

�100,000 —o

SCH 58261 120 (100–140) 2.3 (2.0–2.7) —r

ZM 241385 2,000(1,500–2,700)

0.30 (0.10–0.95) 150,000 (87,000–210,000) —s

MRE 3008F20 �10,000 2,000 (�220) �10,000 —t

* Data from saturation experiments; where confidence limits are missing, they are within 0.15 log units.a Bruns et al., 1986; brain membranes (A1), striatum (A2A); radioligands [3H]CHA (A1), [3H]NECA (A2A).b Lohse et al., 1988; brain membranes (A1), striatum (A2A); radioligands [3H]PIA (A1), [3H]NECA (A2A).c Klotz et al., 1991; brain membranes; radioligand [3H]DPCPX.d Olah et al., 1994; transfected CHO cells; radioligand 125I-AB-MECA.e van Galen et al., 1994; transfected CHO cells; radioligand 125I-APNEA.f Cristalli et al., 1992; brain membranes (A1), striatum (A2A); radioligands [3H]DPCPX (A1), [3H]NECA (A2A).g Hutchison et al., 1989; rat brain; radioligands [3H]CHA (A1), [3H]NECA (A2A). Given are IC50 values; based on the experimental conditions Ki values should be about

0.5–0.7 and 0.9 times the IC50 values for A1 and A2A, respectively.h Cristalli et al., 1995; brain membranes (A1), striatum (A2A); radioligands [3H]CHA (A1), [3H]CGS 21680 (A2A).i Camaioni et al., 1997; brain membranes (A1), striatum (A2A); radioligands [3H]CHA (A1), [3H]CGS 21680 (A2A).j Gallo-Rodriguez et al., 1994; rat brain (A1), rat striatum (A2A), transfected CHO cells (A3); radioligand [3H]PIA (A1), [3H]CGS 21680 (A2A), 125I-AB-MECA (A3).k Kim et al., 1994; rat brain (A1), rat striatum (A2A), transfected CHO cells (A3); radioligand [3H]PIA (A1), [3H]CGS 21680 (A2A), 125I-AB-MECA (A3).l Lohse et al., 1987; brain membranes (A1), striatum (A2A); radioligands [3H]PIA (A1), [3H]NECA (A2A).m Jacobson et al., 1986; rat brain, radioligand [3H]XAC.n Jarvis et al., 1989; rat brain; radioligands [3H]CHA (A1), [3H]CGS 21680 (A2A).o Kim et al., 2000; transfected HEK 293 cells (A1), rat striatum (A2A); radioligands [3H]PIA (A1), [3H]CGS 21680 (A2A).p Sauer et al., 2000; brain cortex (A1), striatum (A2A); radioligand [3H]CHA (A1), [3H]CGS 21680 (A2A).q Williams et al., 1987; brain (A1), striatum (A2A); radioligands [3H]CHA (A1), [3H]NECA (A2A). Given are IC50 values; based on the experimental conditions Ki values

should be about 0.5–0.7 and 0.9 times the IC50 values for A1 and A2A, respectively.r Baraldi et al., 1994; rat brain (A1), rat striatum (A2A); radioligand [3H]CHA (A1), [3H]CGS 21680 (A2A).s Poucher et al., 1995; brain cortex (A1), PC 12 cells (A2A), transfected CHO cells (A3); radioligands [3H]PIA (A1), [3H]NECA (A2A) 125I-AB-MECA (A3). Given are IC50

values; based on the experimental conditions Ki values should be about 0.1, 0.2, and 0.5 times the IC50 values for A1, A2A, and A3, respectively.t Varani et al., 2000; brain cortex (A1), striatum (A2A), transfected CHO cells (A3); radioligands [3H]DPCPX (A1), [3H]SCH 58261 (A2A), 125I-AB-MECA (A3).u Bridges et al., 1988; brain (A1), striatum (A2A); radioligand [3H]CHA (A1), [3H]NECA (A2A); DPMA or PD 125944 is compound 16 in this paper.v Trivedi et al., 1989; brain (A1), striatum (A2A); radioligand [3H]CHA (A1), [3H]NECA (A2A).w Jacobson et al., 1987; brain (A1), radioligand [3H]PIA (A1).

540 FREDHOLM ET AL.

amide) was the first representative of this class, witha Ki value of 17 nM (van Muijlwijk-Koezen et al.,1998). Later, VUF5574 (N-(2-methoxyphenyl)-N-(2-(3-pyridyl)quinazolin-4-yl)urea) proved even more po-tent, with a Ki value of 4 nM, being at least 2500-foldselective versus A1 and A2A receptors (van Muijlwijk-Koezen et al., 2000). One of the most selective com-pounds (for human A3 receptors) is MRE-3008-F20,which is also a useful antagonist radioligand at hu-man A3 receptors (Varani et al., 2000).

Species differences in the affinity of adenosine recep-tor ligands, especially antagonists, have been noted. Forexample, 8-phenylxanthines such as XAC that are selec-tive for A1 receptors in the rat are less selective in thehuman, due to a decrease in the A1 affinity and a con-comitant rise in the A2A receptor affinity. As expectedfrom the structural data (see above), species differencesin pharmacology are most marked for ligands at the A3

receptor. In general, the affinity at A3 receptors of mostxanthines and other classes of antagonists is highlyspecies-dependent, and the affinity at human receptorsis typically �100-fold greater than that at rat receptors.While MRS 1523 is an A3 antagonist of broad applica-bility to various species, both MRS 1220 and MRE-3008-F20 are extremely potent in binding to the human butnot rat A3 receptors and should be used cautiously innonprimate species. In the rat, MRS 1220 is selective forthe A2A receptor. In contrast, the affinity of the A3 ago-nist Cl-IB-MECA typically does not vary beyond an or-der of magnitude between species examined. Neverthe-less, one must caution against receptor classificationbased on pharmacological tools alone, especially in non-mammalian species where the receptors have not beencloned and characterized. Despite the fact that there ismuch interest in mouse adenosine receptors owing to thedevelopment of several mouse strains with targeted de-letions, the information on mouse adenosine receptorpharmacology is deficient. In one study potencies of se-lected agonists and antagonists were virtually identicalat mouse and rat A1 receptors (Maemoto et al., 1997).

IX. Signaling

A. G Protein Coupling

The adenosine A1 and A2 receptors were initially sub-divided on the basis of their inhibiting and stimulatingadenylyl cyclase, respectively (van Calker et al., 1979;Londos et al., 1980a). Indeed, A1 and A2 receptors arecoupled to Gi and Gs proteins, respectively (Table 9). TheA3 receptor is also Gi coupled. In addition there is someevidence that the adenosine receptors may signal viaother G proteins (Tables 1 and 9). However, much of thedata on coupling to other G proteins are from transfec-tion experiments and it is not known if such coupling isphysiologically important. Recently, evidence was pre-sented that the A2A receptor may be coupled to differentG proteins in different areas (Kull et al., 2000a). In mostperipheral tissues, the receptor subtype is coupled to Gs.However, in striatum, where the brain A2A receptors areenriched, Gs is very sparse. Here the dominant G proteinof the class is instead Golf. Indeed, immunoprecipitationexperiments show that A2A receptors and Golf are asso-ciated with each other in the medium-sized spiny neu-rons of striatum.

One adenosine receptor may also be coupled to morethan one G protein. This is common after transfection.Furthermore, endogenous A2B receptors of HEK 293cells, human HMC-1 mast cells and canine BR mast cellsare dually coupled to Gs and Gq (Auchampach et al.,1997; Linden et al., 1999).

B. Second Messengers and Signals

After activation of the G proteins, enzymes and ionchannels are affected as can be predicted from what isknown about G protein signaling (see Tables 1 and 9).Thus, A1 receptors mediate inhibition of adenylyl cy-clase, activation of several types of K�-channels (proba-bly via �,�-subunits), inactivation of N-, P-, and Q-typeCa2� channels, activation of phospholipase C�, etc. Thesame appears to be true for A3 receptors. In CHO cellstransfected with the human A3 adenosine receptor bothadenylyl cyclase inhibition and a Ca2� signal are medi-

TABLE 9G protein coupling of the four adenosine receptor subtypes

Adenosine ReceptorSubtype G Protein Effects of G Protein

Coupling Cellular System Reference

A1 Gi1/2/3 2 cAMP1 IP3/DAG (PLC)1 Arachidonate (PLA2)

General, CHO cellsa Freissmuth et al., 1991; Akbar et al., 1994; Freund et al., 1994;Jockers et al., 1994; Gerwins and Fredholm, 1995a,b

1 PEtOH (PLD) DDT1MF-2Go Jockers et al., 1994; Matovcik et al., 1994

A2A Gs 1 cAMP General Olah, 1997Golf 1 cAMP Kull et al., 2000a; Corvol et al., 2001G15/16 1 IP3 COS-7a Offermanns and Simon, 1995

A2B Gs 1 cAMP General Pierce et al., 1992Gq/11 1 IP3/DAG (PLC) HMC-1, HEK 293a Gao et al., 1999; Linden et al., 1999b

A3 Gi2,3 2 cAMP General, CHO cellsa Palmer et al., 1995Gq/11 1 IP3/DAG (PLC) CHO cellsa Palmer et al., 1995

a Receptor transfected cell system.b References propose Gq/11 coupling without direct evidence for Gq/11 activation.


ated via a Gi/o-dependent pathway (Klotz et al., 2000).Given that many of the steps in the signaling cascadeinvolve signal amplification, it is not surprising that theposition of the dose-response curve for agonists will de-pend on which particular effect is measured. For exam-ple, it was recently found that the so called receptorreserve in DDT1 MF-2 cells appears very different de-pending on whether G protein activation or cAMP accu-mulation is measured (Baker et al., 2000). Both A2A andA2B receptors stimulate the formation of cAMP, butother actions, including mobilization of intracellular cal-cium, have also been described. Actions of adenosine A2Areceptors on neutrophil leukocytes are due in part tocAMP (Fredholm et al., 1996; Fredholm, 1997; Sullivanet al., 2001), but cAMP-independent effects of A2A recep-tor activation in these cells have also been suggested(Cronstein, 1994).

C. Adenosine Receptor-Mediated Changes in CellProliferation and in Mitogen-Activated Protein KinaseActivation

It was shown more than 15 years ago that adenosineA1 and A2B receptors could regulate proliferation anddifferentiation in vascular smooth muscle cells (Jonzonet al., 1985). Since then several examples of such effectshave been described, and they may be related to changesin mitogen-activated protein kinases (MAPK), whichplay an essential role in processes such as cell differen-tiation, survival, proliferation, and death. The family ofMAPK consists of the extracellular regulated kinases(ERK) such as ERK1/2, and the stress-activated proteinkinases (SAPK), such as p38 and jun-N-terminal kinase(JNK). These kinases, usually activated via receptortyrosine kinases (Seger and Krebs, 1995), have also beenshown to be activated by G protein-coupled receptors.

Adenosine A1 receptors transiently expressed inCOS-7 cells can activate ERK1/2 via �,�-subunits re-leased from pertussis toxin-sensitive G proteins Gi/o(Faure et al., 1994). Studies in CHO cells stably express-ing the human A1 receptor later showed that the activa-tion of ERK1/2 by A1 receptors is time- and dose-depen-dent (Schulte and Fredholm, 2000) and sensitive to thephosphoinositol-3-kinase inhibitors wortmannin and LY294002 (Dickenson et al., 1998). Although speculative,this may imply a receptor tyrosine kinase transactiva-tion as described for the epidermal growth factor (Daubet al., 1997).

Activation of A2A receptors also increases MAPK ac-tivity. Adenosine agonists exerting mitogenic effects onhuman endothelial cells via the adenosine A2A receptoractivate ERK1/2 using the cAMP-ras-MEK1 pathway(Sexl et al., 1997). However, the signaling pathwaysused by the A2A receptor seem to vary with the cellularbackground and the signaling machinery that the cellpossesses. Thus, the A2A receptor-mediated ERK1/2 ac-tivation in CHO cells is dependent on Gs-cAMP-PKA-rap1-p68 B-raf-MEK1. On the other hand, the A2A re-

ceptor-mediated activation in HEK 293 cells involvesPKC, ras, and sos, but not Gs, cAMP, or PKA, eventhough cAMP levels do rise in a Gs-dependent manner(Seidel et al., 1999).

A2A receptor activation may not only stimulate, butalso inhibit ERK phosphorylation. Activation of guineapig A2A receptors expressed in CHO cells inhibitedthrombin-induced ERK1/2 activation (Hirano et al.,1996). This inhibition was cAMP- and wortmannin-sen-sitive, implying that the nonselective adenosine analogNECA affects the two distinct pathways leading fromthe thrombin receptor to MAPK. In PC12 cells, activa-tion of endogenously expressed A2A receptors inhibitsnerve growth factor (NGF)-induced ERK1/2 phosphory-lation (Arslan et al., 1997), even though activation ofthese receptors alone (i.e., in the absence of NGF) canlead to an activation (Arslan and Fredholm, 2000).

The adenosine A2B receptor is the only subtype that sofar has been shown to activate not only ERK1/2 but alsoJNK and p38. In human mast cells (HMC), adenosinereceptor activation leads to a time- and dose-dependentactivation of ERK1/2 with a maximal degree of phos-phorylation at 5 min, whereas p38 and JNK show adifferent kinetic profile with maximal phosphorylationat 1 and 10 to 15 min, respectively (Feoktistov et al.,1999). Adenosine A2B receptor-mediated activation ofMAPK is relevant for IL-8 secretion and consequentlyfor mast cell activation. In untransfected HEK 293 cells,a cascade depending on Gq/11, PLC, genistein-insensitivetyrosine kinases, ras, B-raf, and MEK1/2 has been de-lineated (Gao et al., 1999). NECA concentrations used inthese studies of endogenous HEK 293 A2B receptorsrevealed the same potency in activating ERK and ad-enylyl cyclase (EC50 values in the micromolar range)whereas results from another study in transfected cells(Schulte and Fredholm, 2000) show a nearly 100-foldhigher potency of both NECA and adenosine in inducingERK1/2 phosphorylation than in inducing cAMP produc-tion. The EC50 value for ERK1/2 phosphorylation intransfected CHO lies in the nanomolar range, whereascAMP production is half-maximally activated around 1to 5 �M NECA. Thus, a G protein-coupled receptor canhave substantially different potencies on different sig-naling pathways in the same cellular system.

It was recently reported that, in addition to bindingadenosine and adenosine analogs, the A2B receptor incomplex with another protein, DCC (deleted in colorectalcancer), may bind netrin-1, a protein that is involved incontrolling axon elongation, and that netrin effects de-pend on the presence of the A2B receptor (Corset et al.,2000). These results have, however, recently been con-tested (Stein et al., 2001). Nevertheless, it seems possi-ble that the A2B receptors play very important roles incell proliferation and/or differentiation. These effectsmay not only be stimulatory, however. In vascularsmooth muscle cells, activation of A2B receptors stronglydecreases the mitogenic effects of different growth fac-

542 FREDHOLM ET AL.

tors (Jonzon et al., 1985; Dubey et al., 1996, 1998),probably secondary to a blockade of MAP kinases(Dubey et al., 2000) stimulated by these growth factors.

The adenosine A3 receptor has been suggested to ac-tivate ERK1/2 in human fetal astrocytes (Neary et al.,1998). A recent study, indeed, shows a clear activation ofERK1/2 via the human A3 receptor expressed in CHOcells (Schulte and Fredholm, 2000). Both NECA and theendogenous agonist adenosine lead to a time- and dose-dependent increase in ERK1/2 phosphorylation alreadyat concentrations as low as 10 to 30 nM. The A3 receptoragonists Cl-IB-MECA and IB-MECA have been reportedto potently inhibit and less potently to activate apoptosisin various cells (Abbracchio et al., 1997). In RBL-2H3mast-like cells, Cl-IB-MECA potently blocks UV irradi-ation-induced apoptosis by a process that correlates withprotein kinase B phosphorylation and is blocked by per-tussis toxin and wortmannin (Gao et al., 2001).

Thus, the adenosine receptor-mediated activation ofMAPK is similar to that encountered in the remainder ofthe field of GPCR-mediated MAPK activation (Gutkind,1998; Sugden and Clerk, 1998; Luttrell et al., 1999). Thecommon feature of all adenosine receptors, however, isthe positive coupling to ERK1/2 even though the classi-cal cAMP/PKA pathway is both activated (A2) and in-hibited (A1/3). Depending on the cellular background therequired signaling elements vary widely, although acti-vation of one of the small GTP-binding proteins p21ras

and rap1 is essential.

D. Interactions with Other Receptor Systems

Adenosine is believed to play modulatory roles in avariety of tissues and physiological circumstances.Adenosine is, as discussed above, not primarily releasedin a transmitter- or hormone-like fashion, but instead itappears to be formed by groups of cells as part of aresponse, e.g., to challenges in energy metabolism. It canalso be formed by breakdown of ATP released by cellseither in a regulated fashion or in response to massivetrauma. Adenosine is therefore likely to act in concertwith several other messengers (transmitters, hormones,growth factors, autacoids). It is outside the scope of thistype of review to delineate all such interactions and afew examples will have to suffice.

Adenosine can, as noted above, activate phospholipaseC via adenosine A1 receptors and a Gi-dependent mech-anism. Interestingly, adenosine acts synergistically withnucleotides, such as ATP or UTP (Gerwins and Fred-holm, 1992a), histamine (Dickenson and Hill, 1993), orwith bradykinin (Gerwins and Fredholm, 1992b). ATP,histamine, and bradykinin instead act via Gq/11. Theinteraction was believed to involve �,�-subunits releasedfrom Gi proteins by A1 receptors, and �q/11-subunitsderived via the other receptor, somehow acting synergis-tically at the level of PLC. The involvement of �,�-sub-units has been confirmed (Dickenson and Hill, 1998).More recently, good evidence was provided that the

mechanism involved exchange of �,�-subunits betweenthe two G protein pathways (Quitterer and Lohse, 1999),but other mechanisms cannot be excluded. It was alsorecently shown that activation of PLC by Gi-coupledreceptors requires that the enzyme is already activated(Chan et al., 2000), e.g., by ATP. Given that ATP israpidly broken down to adenosine and that ATP andadenosine act synergistically, we may be dealing with abiologically quite important mechanism.

In the brain there is evidence of important interac-tions between adenosine A1, dopamine D1, and NMDAreceptors. First, it is known that activation of NMDAreceptors can increase the release of adenosine (Hoehnet al., 1990; Pedata et al., 1991; Chen et al., 1992; Man-zoni et al., 1994; Delaney et al., 1998; Delaney andGeiger, 1998). Second, it is well known that adenosine,by activating presynaptic receptors, can decrease therelease of glutamate, and thereby the activation ofNMDA receptors (Fredholm and Hedqvist, 1980; Fred-holm and Dunwiddie, 1988; Poli et al., 1991; Dunwiddieand Fredholm, 1997). In addition, activation of adeno-sine A1 receptors reduces the NMDA currents by apostsynaptic action (de Mendonca and Ribeiro, 1993; deMendonca et al., 1995) and reduces toxicity (Finn et al.,1991). A third interaction is that dopamine D1 andNMDA receptors act synergistically in the brain, e.g., onthe expression of immediate early genes (Berretta et al.,1992) and several long-term effects of dopamine D1 ac-tivation can be blocked by antagonizing NMDA recep-tors (Wolf et al., 1994; Konradi et al., 1996). However,this synergistic interaction differs between regions(Keefe and Gerfen, 1996). Part of this enhancement maydepend on the ability of dopamine D1 agonists to phos-phorylate DARPP-32, which in turn acts to decrease thedephosphorylation of NMDA receptors, leading to theirremaining in an active conformation (Blank et al., 1997).Activation of NMDA receptors conversely leads to de-creased DARPP-32 phosphorylation (Halpain et al.,1990). A fourth interaction is between dopamine D1 re-ceptors and adenosine A1 receptors, which have beenshown to act antagonistically (Ferre et al., 1994b, 1998;Popoli et al., 1996). All these interactions may be func-tionally connected (Harvey and Lacey, 1997). Thus, dop-amine D1 activation has been shown to reduce release ofglutamate by enhancing NMDA activity and thereby re-lease of adenosine, which is the agent that acts at presyn-aptic A1 receptors.

Adenosine A2A receptors are highly enriched in thebasal ganglia of several species including human. Therethey are present in highest abundance on a subset of theGABAergic output neurons, namely those that project tothe globus pallidus (Schiffmann et al., 1991; Fink et al.,1992; Svenningsson et al., 1997; Rosin et al., 1998).These neurons also express dopamine D2 receptors andit is abundantly clear that the two receptors interact inbinding assays (Ferre et al., 1991), on signal transduc-tion (Kull et al., 1999), and behaviorally (Fink et al.,


1992; Fredholm et al., 1999; Svenningsson et al., 1999b).It appears that one major role of dopamine in the stria-tum is to suppress signaling via A2A receptors (Sven-ningsson et al., 1998b, 1999a; Chen et al., 2001). Giventhat antagonists of D2 receptors are used in the treat-ment of schizophrenia, there are interesting implica-tions for the pathophysiology of that disease. AdenosineA2A and dopamine D2 receptors also are colocalized onarterial chemoreceptors of the carotid body (Gauda etal., 2000). Carotid sinus nerve activity is augmented byadenosine binding to adenosine A2A receptors and atten-uated by dopamine binding to dopamine D2 receptors.

Adenosine A2B receptors are present on many cells,although in low abundance. It has been repeatedly foundthat signaling via the A2B receptor is strongly influencedby concurrent signaling via other receptors that affectPLC-Ca2�-PKC. However, the type of interaction variesfrom cell to cell. In brain slices the cAMP accumulationmediated by activation of A2B receptors is markedlyenhanced by drugs that stimulate PKC (Hollingsworthet al., 1985; Fredholm et al., 1987b; Nordstedt and Fred-holm, 1987). Since cAMP accumulation in brain slicespredominantly reflects events in glial cells, it seemslikely that this is the target cell for the interaction. Inglial cells the positive effect of PKC activation can, how-ever, be counteracted by an inhibitory effect of strongincreases in intracellular calcium (Altiok et al., 1992;Altiok and Fredholm, 1992, 1993; Balmforth et al.,1992). A similar effect is seen in lymphocytes (Fredholmet al., 1987a; Nordstedt et al., 1987, 1989; Kvanta et al.,1989). In these cells it could be shown that the stimula-tory effect of PKC activation predominantly occurs notat the receptor itself but at a signaling step after thereceptor.

Numerous other examples of interaction betweenadenosine receptors and other receptors have been re-ported, and such interactions are likely to be the rulerather than the exception. This has important conse-quences. For example, if two receptors act synergisti-cally then blockade of either receptor will virtually abol-ish the response. This can easily be erroneouslyinterpreted as evidence that the receptor that is blockedis solely responsible for the response studied. Anotherimportant case should also be mentioned. Sometimesmore than one adenosine receptor is expressed on asingle cell. The resultant response will therefore be amixed one and consequently pharmacology may be quiteatypical.

X. Receptor Regulation

Exposure of any GPCR to agonists for shorter orlonger times generally leads to the attenuation of theagonist response (for recent reviews, see Krupnick andBenovic, 1998; Lefkowitz, 1998; Pitcher et al., 1998;Ferguson, 2001). Adenosine receptors are no exception(Olah and Stiles, 2000). However, the magnitude of this

response and the mechanisms involved seem to varybetween the adenosine receptor subtypes. For example,there are major differences between the two Gi/o coupledreceptors, A1 and A3, when stably transfected into CHOcells (Palmer et al., 1996). Whereas the A1 receptorappears to desensitize slowly and incompletely, the A3receptor desensitizes very rapidly. This difference maybe due to differences in the ability of G protein receptorkinases to phosphorylate the two receptors. However,there may be additional factors to consider. Thus inDDT1 MF-2 cells, which constitutively express A1 recep-tors, desensitization is slow when cyclase responses arestudied (Ramkumar et al., 1991), but have been reportedto be rapid when, instead, phospholipase C activation isexamined (Ciruela et al., 1997). In this case, phosphor-ylation mediated via other kinases might be involved.The receptor has also been reported to internalize withinhalf an hour of agonist stimulation together with aden-osine deaminase (Saura et al., 1996). However, manyquestions remain to be solved in the case of receptorregulation in DDT1 MF-2 cells, and not all reports areconsistent (Olah and Stiles, 2000).

Recently, it was found that agonist treatment canrapidly translocate adenosine A1 receptors out of caveo-lae, providing another type of explanation for alteredreceptor signaling (Lasley et al., 2000). In addition, us-ing still other types of cells or examining the situation invivo, several groups have described a more or less rapiddecrease in receptor number following agonist adminis-tration (Green, 1987; Longabaugh et al., 1989; Green etal., 1990; Fernandez et al., 1996; Hettinger et al., 1998).These changes are not accompanied by changes in themRNA. Furthermore, antagonist treatment has longbeen known to up-regulate adenosine A1 receptors(Fredholm, 1982), even though other adenosine recep-tors are unaffected. Thus, we still do not understand thefactors that are important in regulating adenosine A1and A3 receptors under physiological and pathophysio-logical conditions.

Relatively little is known about desensitization of A2Breceptors, except that desensitization does occur in dif-ferent cell lines (Mundell and Kelly, 1998; Peters et al.,1998; Haynes et al., 1999). The mechanism may involveG protein receptor kinases. A2A receptors coupled to Gsproteins are believed to desensitize rapidly (Ramkumaret al., 1991; Chern et al., 1993, 1995; Palmer and Stiles,1997). Several mechanisms, including phosphorylationof Thr298 via receptor kinases, and phosphorylation viacAMP-dependent kinases have been implicated. Never-theless, there is good evidence that under in vivo condi-tions A2A receptors on leukocytes can be tonically acti-vated by endogenous adenosine (Cronstein, 1994;Fredholm, 1997). Similarly, there is excellent evidencethat A2A receptors on striatopallidal neurons are toni-cally activated by endogenous adenosine (Svenningssonet al., 1995, 1999a, 2000). Conversely, when tonic aden-osine A2A receptor activation is unopposed by dopamine

544 FREDHOLM ET AL.

for a long period of time, the response to agonist stimu-lation decreases (Zahniser et al., 2000), even thoughsignificant effects remain (Chen et al., 2001). This is notdue to any change in the receptor number, however.Thus, desensitization of adenosine A2A receptors, viawhatever mechanism, may be important mainly underexperimental or pathological situations.

XI. Assay Systems

Adenosine receptors and their ligands may be inves-tigated in binding and functional assays. The first usefuladenosine analogs R-PIA and NECA were developedusing assays in intact animals. Specifically, blood pres-sure, heart rate, and body temperature were some of thereadouts (Vapaatalo et al., 1975; Raberger, 1979). Stud-ies using changes in cAMP in fat cells, liver, Leydig cells,brain homogenates, and cultured brain cells were in-strumental in the definition of A1 (Ri) and A2 (Rs) recep-tors (van Calker et al., 1978, 1979; Londos et al., 1980b).Binding assays became available in the early 1980s, butfunctional assays continued to play an important role.For example, studies on coronary blood flow in dogs wereused to screen and develop a large number of compoundsacting at A2A receptors (Daly et al., 1986). Even todayseveral functional assays remain very useful, not leastbecause they examine receptors associated with the nat-ural signaling pathways. For example, A1 receptor-me-diated inhibition of adenylyl cyclase can be studied in fatcells (Fredholm, 1978; Londos et al., 1978; Longabaughet al., 1989) whereas platelet membranes is a preferredsystem for stimulation of adenylyl cyclase via A2A aden-osine receptors (Klotz et al., 1985). A2B receptors havebeen examined using fibroblasts (Bruns, 1980, 1981)and rat aorta (Collis and Brown, 1983; Poucher et al.,1995).

For a long time rat brain membranes were the stan-dard system for examining binding to A1 (whole brain;Bruns et al., 1980; Schwabe and Trost, 1980) and A2Areceptors (striatum; Bruns et al., 1986). Now cell linesstably transfected with the receptor subtype of interesthave become the standard systems for the characteriza-tion of ligands, as they have several major advantages(Klotz et al., 1998). Transfected cells allow for the com-parative characterization of receptor subtypes from dif-ferent species including humans. With the expression ofsingle receptor subtypes, cross-reaction of nonselectiveligands with receptors not under investigation does notoccur. In addition, receptors like the A3 subtype thatoccur naturally only at low expression levels becomeavailable for in vitro studies. Cellular models with trans-fected receptors may also be used for the characteriza-tion of effector coupling. All the typical second messen-ger responses for the adenosine receptor subtypes havebeen identified in CHO cells stably transfected with thehuman subtypes. However, these results may not repre-sent the physiological situation because the receptor

density and the availability of G proteins and othercomponents necessary for signal generation may be dif-ferent in artificial cellular models.

XII. Physiological Roles—Therapeutic Potential

Recently, genetically modified mice have been gener-ated that provide insights into the physiology and patho-physiology of the different adenosine receptors. Theadenosine A2A receptor was first knocked out. The groupin Brussels that first cloned the adenosine receptors alsogenerated the first knock-out mice, in which the firstcoding exon of the A2A receptor was targeted (Ledent etal., 1997). Later a group in Boston targeted the secondexon (Chen et al., 1999). Using these mice has shownthat A2A receptors play a role in mediating pain viaperipheral sites, inhibiting platelet aggregation and reg-ulating blood pressure (Ledent et al., 1997). A2A recep-tors are also critically important for the motor stimulanteffects of caffeine (Ledent et al., 1997; El Yacoubi et al.,2000). It has also been demonstrated that A2A receptorscontribute to ischemic brain damage in adult mice (Chenet al., 1999).

Mice with a targeted disruption of the A3 receptorshow a decreased effect of adenosine analogs on mastcell degranulation (Salvatore et al., 2000) and a conse-quent decrease in vascular permeability (Tilley et al.,2000). These animals also, surprisingly, show increasedcardiovascular effects of administered adenosine (Zhaoet al., 2000). Given the species differences in A3 receptordistribution and pharmacology it is, however, not clearthat the roles of this receptor are similar in humans.

Mice with a targeted disruption of A1 receptors havealso been generated (Johansson et al., 2001). These miceshow increased anxiety and are hyperalgesic, indicatinga role for A1 receptors in mediating endogenous antino-ciception. In these animals, the effect of adenosine onexcitatory neurotransmission is totally eliminated (Dun-widdie et al., 2000), and the neuronal response to hyp-oxia is markedly altered. They also lack tubuloglomeru-lar feedback and have elevated renin levels (Brown etal., 2001; Sun et al., 2001). Interestingly, all these miceshow essentially normal viability and fertility.

Adenosine protects tissues from ischemic damage ine.g., brain and heart through multiple receptor subtypes(Lasley et al., 1990; Marangos, 1990; Rudolphi et al.,1992a,b; Auchampach and Gross, 1993; Deckert andGleiter, 1994; Fredholm, 1996). A1 and possibly A3 re-ceptor activation produces preconditioning to protect theheart and other tissues from subsequent ischemic in-jury. Rapid preconditioning is mediated by a pathwayincluding PKC and increased mitochondrial K-ATPchannel activation (Sato et al., 2000). A late phase ofpreconditioning due to A1 receptor activation in the rab-bit heart is mediated in part by the induction of manga-nese superoxide dismutase (Dana et al., 2000).


Fishman and coworkers demonstrated that low dosesof adenosine cause a strong inhibition of lymphoma cellproliferation. A similar effect was seen with low doses ofIB-MECA, a somewhat selective agonist for A3 recep-tors, whereas no such action was observed with theselective A1 receptor agonist CCPA (Fishman et al.,2000b). The same research group showed that adenosinealso acts as a chemoprotective agent by stimulatinggranulocyte-colony-stimulating factor production. Inthis case, a combined action via both A1 and A3 receptorsappeared causal (Fishman et al., 2000a).

Activation of A2A receptors protects tissues from in-jury by reducing inflammation during reperfusion fol-lowing ischemia. CGS 21680 inhibits neutrophil accu-mulation and protects the heart from reperfusion injury(Jordan et al., 1997). However, similar cardiac protec-tion has recently been attributed to A3 receptor activa-tion (Jordan et al., 1999).

The localization of A2A receptors to the dopamine richareas of the brain and the behavioral effects of methyl-xanthines suggested that antagonists at A2A receptorsmight be useful as adjuvants to dopaminergic drugs inParkinson’s disease (Fredholm et al., 1976; Ongini andFredholm, 1996; Ferre et al., 1997; Svenningsson et al.,1999b; Impagnatiello et al., 2000) as well as schizophre-nia (Ferre et al., 1994a; Rimondini et al., 1997). Thisidea has gained additional support by the demonstrationthat the adenosine A2A receptor exerts effects that are atleast to some extent independent of dopamine acting atD2 receptors (Svenningsson et al., 1995, 1998a, 1999a,2000; Chen et al., 2001). The effect of an A2A receptorantagonist is synergistic with dopamine receptor ago-nists in primate models of Parkinson’s disease (Kanda etal., 2000). Rapid tolerance develops to the motor stimu-lant effects of caffeine, but no such tolerance is seenfollowing long-term treatment with selective adenosineA2A receptor antagonists (Halldner et al., 2000; Popoli etal., 2000; Pinna et al., 2001).

There is evidence that IgE antibodies and mast cellsplay a central role in the symptoms and pathology ofasthma. Aerosolized adenosine has the effect of causingmast cell-dependent bronchoconstriction in asthmaticsubjects, but causes bronchodilation in nonasthmatics(Cushley et al., 1983; Vilsvik et al., 1990). Moreover, thenonselective adenosine receptor antagonist theophyllineis widely used as an antiasthmatic drug although itsmechanism of action is uncertain. A related xanthine,enprofylline (3-propylxanthine) (Robeva et al., 1996; Ja-cobson et al., 1999), is also therapeutically efficacious inthe treatment of asthma, and was thought to actthrough a nonadenosine receptor-mediated mechanismdue to its low affinity at A1 and A2A receptors.

Recently, attention has shifted to the A2B and A3receptor subtypes found on mast cells that, when acti-vated, facilitate antigen-mediated mast cell degranula-tion. The adenosine A3 receptor was initially implicatedas the receptor subtype that triggers the degranulation

of rat RBL 2H3 mast-like cells (Ramkumar et al., 1993)and perivascular mast cells of the hamster cheek pouch(Jin et al., 1997). There is also evidence of mast celldegranulation when agonists of A3 receptors are admin-istered to rats or mice (Fozard et al., 1996; Tilley et al.,2000). In contrast, the A2B receptor has been implicatedas the receptor subtype that facilitates the release ofallergic mediators from canine and human HMC-1 mas-tocytoma cells (Feoktistov and Biaggioni, 1995; Aucham-pach et al., 1997). A role for A2B receptors in humanasthma is also suggested by the efficacy of enprofylline,which at therapeutic concentrations of 20 to 50 �M, onlyblocks the A2B receptor subtype (Fredholm and Persson,1982; Linden et al., 1999). In sum, the literature indi-cates that the release of allergic mediators from mastcells is mediated by A3 receptors and/or A2B receptors.This may result from tissue difference and/or speciesdifferences, with rodent (rat, guinea pig, and mouse)responding primarily to A3, and canine or human mastcells mainly to A2B adenosine receptor stimulation.

It is remarkable that despite intensive efforts, rela-tively few adenosine receptor ligands have made it intoclinical trials. Adenosine itself is being marketed in sev-eral countries, for two purposes. Adenocard (i.v.) re-stores normal heart rhythm in patients with abnormallyrapid heartbeats originating in the upper chambers ofthe heart, so-called paroxysmal supraventricular tachy-cardia. Adenoscan (i.v.) is indicated as an adjunct tothallium cardiac imaging in the evaluation of coronaryartery disease in patients unable to exercise adequately.In the 1970s, metabolically stable adenosine receptoragonists were tested clinically as antihypertensives. Inthe last decade GR79236 (N-[(1S, trans)-2-hydroxycyclo-pentyl]adenosine) has been tested in human volunteersfor various indications such as adjuvant therapy in in-sulin resistance (type II diabetes) and, more recently, inprimary headache (P. P. A. Humphrey, personal commu-nication).

With respect to antagonists, it can be argued thatcaffeine, the most widely used psychotropic substance,has its main action via adenosine receptors. The samemight hold true for theophylline and enprofylline, bothused in the treatment of asthma. Currently, selectiveadenosine A1 receptor antagonists are undergoingclinical trials. A recently concluded Phase II trial withBG9719 ((S)-1,3-dipropyl-8-[2-(5,6-epoxynorbornyl)]xanthine) was successful in treating acute renal fail-ure in patients with congestive heart failure. This wasseen as a critical factor for future development of asecond-generation product, BG 9928. The A2A adeno-sine antagonist KW6002 (1,3-diethyl-8-(3,4-dime-thoxystyryl)-7-ethylxanthine) is undergoing clinicaltrials as an anti-Parkinson agent (F. Suzuki, personalcommunication). Other compounds of this class arealso under development. Thus, adenosine receptorsremain an attractive target for drug development.

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